The present invention belongs to the biotechnology field and relates to a thermophilic alkaline phosphatase (or phosphoesterase).
The alkaline phosphatase is an important enzyme, which is widely distributed in various organisms, and participates in cellular phosphorus metabolism. The alkaline phosphatase is a non-specific phosphomonoesterase, which produces a phosphoserine intermediate and finally produces inorganic phosphorus and alcohol. The amino acid sequences and the corresponding genes of the alkaline phosphatase have been obtained from many prokaryote and eukaryote, such as E.Coli, B. subtilis, yeast, calf intestine, human placenta, etc. (J Bio Chem. 1991, 266: 1077-84).
The alkaline phosphatase is an important enzyme tool in the molecular biology study. It can be used in dephosphorization of the termini of DNA or RNA fragment in gene cloning, as a reagent for enzyme-linked assay in immunology research, and as a label for nucleic acid hybridization or detection of the PCR products.
Nucleic acid hybridization, one of the most extensive applied techniques in molecular biology, is a technique that detects the complementary nucleotide sequences by using the labeled DNA or RNA fragment as a probe. Typically, the label of the nucleotide probe is an isotope, such as 32P or 35S. Though the isotope label is very sensitive, its conventional biological and medical application and commercial kits are substantially restricted by the short half-life, the danger to the operator during the manipulating procedure, and the trouble of dealing with the isotopic wastes, etc. People have extensively studied the labeling of the nucleotide probe with the non-isotopic materials during the last decade (Mattews J. Anal Biochem.1988, 169:1-25).
For the time being, the common labels include enzyme, fluorescein, biotin, digoxin (Europe Patent EP 304934). The labeling methods can be divided into direct and indirect techniques based on whether the label can be detected directly or not after hybridization. The major indirect labels are haptens, such as biotin and digoxin; and the major direct labels are enzymes and fluoresceines. The alkaline phosphatase is the most extensively applied enzyme in both direct and indirect labeling methods. In the 1980""s, the direct nucleotides labeling using the alkaline phosphatase was reported (Jablonski E: Nucleic Acid Res. 1986, 14: 6115xcx9c6128). The enzymes described in the reports were mainly calf intestine alkaline phosphatase and E.Coli alkaline phosphatase. These alkaline phosphatases have a main drawback of being instable under high temperature, thus not suitable for hybridization in higher temperature. However, the hybridization under higher temperature is usually beneficial for reducing the background and enhancing the specificity. Additionally, these enzymes can not tolerate the strong hybridization and elution conditions, such as high concentration of SDS. Because of the poor thermostability, the oligonucleotides directly labeled by these alkaline phosphatases can not be used as the primers for the polymerase chain reaction (PCR).
The thermophilic bacteria are microorganisms that can live and grow at more than 55xc2x0 C. Most enzymes in thermophilic bacteria, such as the thermophilic DNA polymerase used extensively in PCR, are thermophilic enzymes that have high application value. But there was no report or patent about the alkaline phosphatase from thermophilic bacteria before the present invention.
The present invention provides an alkaline phosphoesterase which has higher thermostability and is suitable for extensive use.
As used in this invention, the term xe2x80x9cthermophilic alkaline phosphatasexe2x80x9d (xe2x80x9cFD-TAPxe2x80x9d for short) means the enzyme with the following features or characteristics: its optimum reaction temperature is above 50xc2x0 C., and its enzyme activity remains at least 70% after the incubation at 70xc2x0 C. for 30 mins. As far as the same enzyme is concerned, the features or characteristics described above are observed under optimal preservation conditions and reaction systems. The features or characteristics might fluctuate as the conditions or reaction systems change.
The present invention provides a thermophilic alkaline phosphatase that is homologous or substantially homologous to the amino acid sequence shown in Table 1 (SEQ ID NO:2).
The signal peptide composed of 26 amino acid residues is underlined at the N-terminus of the amino acid sequence.
The present invention further provides DNA fragments which are homologous or substantially homologous to the nucleotide sequence as shown in Table 2 which encodes the enzyme of the invention.
As used herein, the phrase xe2x80x9cDNA fragment(s)xe2x80x9d described above includes single- or double-stranded DNA.
Based on the specific circumstance, the term xe2x80x9chomologousxe2x80x9d means that (1) a DNA fragment has the identical nucleotide sequence when comparing with another DNA fragment; or (2) a protein has the identical amino acid sequence when comparing with another protein.
The term xe2x80x9csubstantially homologousxe2x80x9d means that: (1) compared with another DNA fragment, a DNA fragment has enough identical nucleotide sequence so that the translated protein has the same or similar features or characteristics; (2) compared with another protein, a protein has enough identical amino acid sequence, so that both proteins have the same or similar features or characteristics.
There are various organisms, such as prokaryote, yeast and mammals, which can be used as the resources for the thermophilic alkaline phosphatase or its DNA. Preferably, said organism is a prokaryote, especially the thermophilic bacteria, such as the commercially available bacteria Thermus thermophilus. 
One can make the DNA fragment encoding the thermophilic alkaline phosphatase partially deleted by using the genetic engineering techniques. Serial deletions can be made from 5xe2x80x2 terminus to 3xe2x80x2 terminus or from 3xe2x80x2 terminus to 5xe2x80x2 terminus of the DNA fragment. Alternatively, the DNA fragment can be deleted sequentially from both ends to the center. In addition, the middle part of the DNA fragment can be deleted and the two end parts can then be ligated together. The shortest DNA fragment is composed of only 60 bases after deletion. Generally, the polypeptide encoded by the deleted DNA fragment retains the features or characteristics of the thermophilic alkaline phosphatase.
The present invention also provides a recombinant vector which comprises one or more copies of the DNA fragment (or the deleted DNA fragment) of the invention. Said vector can be used to express the thermophilic alkaline phosphatase in host cells.
The vector includes eucaryotic vector and prokaryotic vector, preferably the prokaryotic vector so as to facilitate the amplification in prokaryote. The prokaryotic vector includes bacteriophage xcex (such as xcexgtt11, xcexDash, xcexZapII, etc.) and plasmid (such as pBR322, pUC series, pBluescript, etc.). Plasmid is preferred. The above vectors are commercially available.
The present invention also provides a microorganism transformed by the recombinant vector of the invention. Gram-negative bacteria, especially E.Coli, are first recommended to be used as the host cells.
The recombinant vector of the invention can be obtained by using the following protocol:
(1) isolating the chromosome DNA from the prokaryotic organism, and digesting said DNA with an appropriate restriction endonuclease;
(2) integrating the digested DNA into a vector, using said recombinant vector to transform an appropriate host, and then constructing the gene library;
(3) screening the gene library of step (2) using an appropriate method;
(4) analyzing the positive clone screened out in step (3).
The chromosome DNA can be isolated by treating the prokaryote cells with lysozyme and then adding proteinase K.
The DNA can be digested using an appropriate restriction endonuclease according to the conventional molecular biology methods known in the art. The digested DNA is ligated into an appropriate clone vector, and the recombinant vector is used to transform appropriate organism to construct the gene library. The detailed description about these protocols can be found in laboratory manuals of gene engineering (Sambrook J, et al. In: Molecular Cloning, A Laboratory Manual. 2 ed., CSH Press, 1989).
The gene can be isolated by screening a library using the following methods: (A) hybridization using oligonucleotide probe; (B) polymerase chain reaction (PCR); (C) screening with a specific antibody; and (D) screening based on enzymatic activity. In situ hybridization with nucleic acid or oligonucleotide probes is a common method, however, screening based on enzymatic activity is preferred in the invention because it is easy to detect the activity of the thermophilic alkaline phosphatase. Moreover, the positive clones, which express the thermophilic alkaline phosphatase, can be screened in situ from colonies based on its thermostability.
The inserted DNA fragments in the positive clones can be bi-directionally sequenced by Sanger dideoxy-mediated chain termination method (conventional radioactive isotope manual sequencing or automatic sequencing by the automatic sequencing apparatus). The result is shown in FIG. 1.
The present invention also includes a method for producing the thermophilic alkaline phosphatase that is homologous or substantially homologous to the amino acid sequence shown in FIG. 1, which comprises:
(1) transforming the appropriate host cells with the DNA fragment (or partially delected DNA fragment) encoding the enzyme of the invention or with a recombinant vector containing said DNA fragment;
(2) culturing the transformed host cells in the appropriate medium;
(3) isolating and purifying the protein from the cultured medium or the host cells.
The recombinant thermophilic alkaline phosphatase can be expressed under the control of any appropriate promoters and translation regulatory elements. The suitable hosts include the prokaryote, yeast, mammal cells, insect and plant, etc. The prokaryote is preferred and E.Coli is more preferred. The selection of vector depends on the particular host. In E.Coli, plasmids, such as pJLA503 (Lehauder B, et al. Gene, 1987; 53: 279-283) and pET series (a product available from Stratagene), are commonly used as the expression vector. Typically, the culture medium for E.Coli is abundant medium, such as 2xc3x97YT, etc. Based on the different vectors, the proteinase expression can be induced by changing the temperature or using a chemical method (such as using ITPG).
The thermophilic alkaline phosphatase can be isolated and purified from the cultured cells or medium. If the expressed enzyme protein is present inside the cells, the cells are centrifuged, then lysated by ultrasonic wave, lysozyme, or frozen-thaw cycles. The raw products can be obtained by centrifuging and filtering the cell lysate. If the enzyme protein is secreted into the medium, the enzyme can be obtained by centrifuging and removing the cells, and then purifying from the supernatant. There are many methods for the purification of the enzyme, such as salting-out, ultra-filtration, dialysis, ion-exchange chromatography, HPLC, etc. During the purification of the enzymes, the contaminated proteins can be eliminated effectively by incubating in an elevated temperature (such as 60xc2x0 C.) for a period of time, which makes the purification procedure easier and more convenient.
The thermophilic alkaline phosphatase is useful for the labeling of nucleotides, proteins, or other biomacromolecules, and dephosphorizing the termini of DNA or RNA fragments in gene cloning. The primary use is to label nucleic acids or oligonucleotides directly. There are three main applications of labeled nucleic acids or oligonucleotides: (1) they can be used as the probes for nucleic acid hybridization and foot printing assay, including Southern blot, Northern blot, Slot blot, dot blot, Southern-western blot, hybridization in situ, etc.; (2) they can be used as the primers for nucleic acid amplification in vitro; (3) they can be used for DNA sequence analysis. The linkage between the enzyme protein and nucleic acids or oligonucleotides is a covalent bond established chemically or physically. The terminal group of nucleic acids or oligonucleotides can be linked to the amino group or hydrosulfide group of enzyme protein by a linker arm. (Ruth et al: DNA 1985; 4:93). The detection methods can be chemical, physical or biological methods. Depending on the solid-phase hybridization or the liquid-phase hybridization, the color visualization method using BCIP/NBT as a substrate or the chemiluminescent method using AMPPD as a substrate (Schaap A, et al. Clin Chem 1989, 35: 1863-1864) are preferred for the solid-phase hybridization; and pNPP is preferred as the substrate for liquid-phase hybridization. A quantitative assay may also be performed.